Project Summary Developmental defects in eye structure commonly account for visual impairment in newborns. One such defect, uveal coloboma, is caused by failed development of the optic fissure and is a significant cause of pediatric blindness worldwide. One pathway central to this process is the Hedgehog (Hh) signaling pathway: loss-of-function mutations in the Hh receptor ptch2, which produce overactive Hh signaling, can result in coloboma. In our recent publication (Gordon and Lusk et al., 2018), using zebrafish, we determined the cellular basis of normal optic fissure formation, and identified specific defects in optic fissure and stalk formation in ptch2 mutants, by utilizing timelapse imaging, 4-dimensional cell tracking, and quantitative analysis of individual cell behaviors. We determined through transplantation experiments that both cell-intrinsic and intercellular signaling molecules are involved in the ptch2 mutant phenotype and that this occurs through a Gli-dependent mechanism. I hypothesize that overactivation of Hh signaling results in the upregulation of transcriptional targets that directly disrupt cell movements and morphogenesis, leading to coloboma. The goal of the work proposed here is to identify these targets and dissect their role in the etiology of coloboma. I propose to use a targeted candidate approach in parallel with an unbiased sequencing approach in an effort to identify factors responsible for the ptch2 mutant coloboma phenotype. Based on my preliminary data, I am investigating Netrin as a cell-extrinsic downstream effector of Hh signaling. In Aim 1, I will dissect the genetic interaction between Netrin and Hedgehog signaling during optic fissure and stalk formation, using gain- and loss-of-function approaches. Because I hypothesize at least one intrinsically-acting molecule downstream of Hh signaling is involved in causing coloboma, in Aim 2, I will use unbiased single-cell RNA-sequencing and functional analysis to identify Hh downstream genes that are responsible for the ptch2 mutant phenotype. I will identify specific transcriptomes from cells responding to overactive Hh signaling and directly test the role of upregulated genes in optic fissure and stalk formation. The work proposed here will allow me to uncover molecular pathways and potentially novel therapeutic targets regulating formation of the optic fissure and stalk, with the ultimate goal being a mechanistic understanding of coloboma.